Gently does it: Protein complexes by paper spray ionisation

Ezine

Published: Feb 1, 2014

Author: Steve Down

Channels: Base Peak

Soft and gentle

In the natural world, many proteins exist as loosely bound complexes with other proteins and biomolecules like RNA and lipids but they are difficult to study intact once they are removed from their own environment. Mass spectrometry is one of the few techniques which can be deployed successfully but it took the emergence of soft techniques like electrospray ionisation to allow unbroken protein complexes to be observed.

Now, US researchers have demonstrated that one of the new generation of soft ionisation mass spectrometry techniques is also sufficiently gentle to analyse noncovalent complexes of proteins without breaking them apart. Vicki Wysocki, Yun Zhang, Yue Ju and Chengsi Huang from The Ohio State University in Columbus turned to paper spray ionisation, one of the new types of ambient mass spectrometry techniques, so called because they take place in the open air.

They are characterised by their simplicity and ease of operation, often by analysing the sample itself with no preliminary sample preparation. For paper spray, a minimal amount of preparation is required, simply making up a solution of the sample. This is loaded onto a small piece of paper with a sharp point which is placed in front of the mass spectrometer. When a high voltage is applied to the paper, ions are formed from the sample and are drawn through the inlet of the spectrometer for analysis.

The technique is gentle, producing protonated or deprotonated molecules, depending on the polarity, without fragmentation. This leads directly to molecular mass information for the analytes and the results are obtained within seconds. So, Wysocki was keen to see if noncovalent complexes, which are often bound together weakly, could survive paper spray ionisation.

Intact protein complexes

The attempts were carried out on four protein complexes which were dissolved in ammonium acetate and dropped onto a small triangle of filter paper about 6 mm across the base and 6 mm high. After comparing two types of commercial filter paper, one was preferred because it contained lower concentrations of trace elements like calcium, sodium and chlorine. These anions and cations stabilise proteins in the gas phase, causing peak broadening.

When a voltage of 2.5-3 kV was applied to the paper through the alligator clip that held it in position, signals were detected in the mass spectrometer for up to 15 minutes. The spectra obtained were very similar to those obtained by nanoelectrospray ionisation, in which it is already known that these fragile noncovalent complexes can survive.

For instance, the paper spray mass spectra of the transthyretin tetramer displayed multiply charged ions from +13 to +15, leading to a molecular mass of 56,170 ± 50 Da. This was close to the 55,746 ± 36 Da extrapolated from the nanoelectrospray spectra. The spectra of the dimer and tetramer of concanavalin A, the dodecamer of diformyl antitryptophan binding RNA attenuation protein, and the pentamer and decamer of human serum amyloid P also closely matched their nanoelectrospray spectra. So, it appears that the weak bonds in the noncovalent complexes survive the ionisation and transport processes.

Despite this, there were some subtle differences, illustrated by the fact that the masses calculated from the charge states of the complexes were always higher for the paper spray spectra. Wysocki attributed this difference to higher residual solvation of the complexes during paper spray ionisation. This was supported by the fact that an increase in the source temperature produced narrower peaks, more like the nanospray spectra.

The conclusive piece of proof that the intact complexes made it into the mass spectrometer was provided by the collision cross sections of the ionised species when they were measured in the attached ion mobility mass spectrometer. The CCSs were the same for complexes ionised by paper spray or nanoelectrospray.

Clinical possibilities

The research team also analysed the human haemoglobin protein complex present in blood. The paper spray mass spectrum was very similar to that of a haemoglobin standard. In addition, the molecular masses of the dimer and tetramer complexes were slightly higher than those from nanoelectrospray, presumably due to insufficient desolvation once again.

The structure of the haemoglobin tetramer was confirmed by tandem mass spectrometry measurements which produced peaks corresponding to the iron-haem group and the haemoglobin α and β monomers and trimers.

This is the first time that paper spray ionisation has been used to detect intact noncovalent protein complexes and it has implications for clinical diagnostics. The ease of operation and ability to support a multiplex platform make it an attractive proposition, once the desolvation problem has been addressed. That will be the goal of future work.